Carbohydrates play a number of extremely important roles in the functioning of living organisms. In addition to their metabolic and storage roles, carbohydrates are covalently attached to numerous other molecules such as proteins and lipids. Molecules such as glycoproteins and glycolipids are generally referred to as glycoconjugates. The biological importance of the carbohydrate portion of glycoconjugates can be seen, for example, in the role the carbohydrate portions play in affecting the ability of glycoproteins to perform their biological functions, including such functions as ligand or receptor recognition.
Carbohydrates display an enormous amount of structural variation. This variation stems not only from the wide variety of available monosaccharide units that may be combined to form a larger polysaccharide or oligosaccharide, but from the multitude of possible structural linkages between monosaccharide units. For example, while an oligopeptide containing three different amino acids has six possible configurations, an oligosaccharide containing three different hexose monomers can form at least 200 possible structures. These molecules may vary enormously with respect to their biological properties. Thus it is of interest to provide methods for analyzing both the identity of the monosaccharides units in a given polysaccharide, and the structure of the linkages joining the monosaccharide units to one another.
A widely used method of analyzing the linkages between various monosaccharides sub-units of a complex carbohydrate is methylation analysis (often referred to as permethylation analysis) of the polysaccharide. Methylation analysis typically involves the methylation of the free hydroxyl groups on a polysaccharide. Since the hydroxyl groups participating in linkages between polysaccharides are not free for methylation, methylation of the polysaccharide reveals the nature of the linkages between monosaccharide units. After a polysaccharide has been methylated, the methylated polysaccharide is then hydrolyzed into its constituent monosaccharide units. The methylated carbohydrate monomers are then separated from each other by gas-liquid chromatography. The structure of the separated monosaccharide units is then identified by mass spectrometry.
Techniques for determining carbohydrate structures by methylation analysis are well established. See, for example, Analysis of Carbohydrates by GLC and MS, Chapter 9, editors Biermann and McGinnis, CRC Press (1988); Lindberg and Lonngren, Methods in Enzyomology Vol. 138, Academic Press (1978); Lavery and Hakomori, Methods In Enzymology Vol. 138, Academic Press (1987); Akhrem, et al., Biochiamica et Biophysica Acta, 714:177-180 (1982); Geyer, et al., Analytical Biochemistry 121:263-274 (1982); Lowe and Nilsson, Analytical Biochemistry, 136:187-191 (1984); Hakomori, J. Biochem., 35:205 (1964); Ciucanu and Kerek, Carbohydrate Research, 151:209-217 (1984); Oakley, et al., J. Carbohydrate Chemistry, 4:53-65 (1985); Bjornadal, et al. Angw. Chem. Int. Ed., 9:610 (1970); Neves, et al., Carbohydrate Research, 152:1-6 (1986).
Methylation analysis of carbohydrates has traditionally required large and expensive pieces of laboratory equipment, e.g., a mass spectrometer, and large quantities, e.g., several milligrams, of carbohydrate for analysis. Thus it is of interest to provide new methods of carbohydrates structural analysis employing comparatively low cost equipment and capable of providing useful data with considerably smaller sample sizes.